Heteroresistance, the simultaneous occurrence of drug resistant subpopulations in an otherwise drug susceptible bacterial population in a patient sample, has created uncertainty in the treatment and diagnosis of tuberculosis (TB) and is thought to be an important driver of multi-drug resistance in Mycobacterium tuberculosis (Mtb) which is a significant threat to global TB control given its broadening distribution and the emergence of what is being called “totally drug-resistant TB” (TDR-TB). It has been well-documented that heteroresistance creates difficulties in the interpretation of rapid molecular drug resistance tests because it leads to ‘indeterminate’ test results, but the clinical significance of heteroresistance is still being evaluated. Previously undetectable levels of Mtb resistant subpopulations within a larger population of susceptible pathogens (i.e., heteroresistance) may be present in as many as 20% of TB patients, and might help explain some of the inconsistency observed in TB treatment outcomes. Previously, there was no accurate means of quantifying heteroresistance dynamics or even detecting the presence of heteroresistance until the resistant sub-population had expanded to >1% of the pathogen population within a clinical sample. At that point, however, the patient's infection was determined to be resistant for the purposes of treatment and a potential window of opportunity for preventing resistance was closed.
The current gold standard method for Mtb drug susceptibility testing (DST) can be used to detect later stages of heteroresistance. This phenotypic test uses a culture-based, indirect proportion method which relies on the detection of growth of >1% of the inoculum on culture medium containing a critical concentration of an anti-TB drug. It is estimated that for every 107-8 Mtb bacilli there is at least one bacillus that has a genetic mutation that renders it naturally resistant to a particular drug. Suboptimal antibiotic treatment causes the few Mtb naturally resistant mutants (by definition, a certain level of heteroresistance) to gain a competitive advantage and subsequently dominate the lesion. The 1% population component of resistant organisms that is detectable by the DST method is well above naturally occurring Mtb resistance mutation rates and, again, detections at this level are typically too late to prevent treatment failure.
Current diagnostic methods are limited in their use and efficacy. The threshold of in vitro Mycobacteria Growth Indicator Tube (MGIT) heteroresistance detection is approximately 1% of the population, but is qualitative and takes weeks to complete. Current molecular tools that detect resistance-conferring mutations in Mtb coding genes and promoter regions which also have the potential to rapidly detect heteroresistance, include DNA sequencing (Sanger and pyrosequencing) and allele specific PCR analysis, but these methods are not sensitive or have limited gene coverage. Sanger sequencing and pyrosequencing each have significant technical limitations for analyzing mixed populations. Sanger sequencing has a well-established detection threshold of ˜25% minor component in a mixed sample and relies on subjective visual evaluation of the electropherogram for “quantification”. Pyrosequencing (e.g., Qiagen Pyromark) is capable of true quantitative sequencing, but has a reported quantification threshold of detection equal to 2.5%-5% of an Mtb population and has limited sequencing depth capability. The PCR-hybridization approach of the line-probe assay has a described sensitivity of detection of a minor resistant variant detection at 5%. Detection of heteroresistance, even at the 1% level provided by MGIT-DST, is insufficient and likely too late to prevent treatment failure. A quantitative and more sensitive method is highly desirable.
Mtb heteroresistance is not a rare phenomenon, occurring in 9-30% of Mtb populations studied, and has been identified in Mtb populations with phenotypic resistance to first line-drugs (INH, RIF, ETH, and STR) and second-line fluoroquinolones (ofloxacin-OFX) and injectables (AMK). It is highly likely that drug resistant organisms are present in most TB lesions, even as very minor population components, given the high bacilli loads that are typically found in patients.
There is also a critical need to detect minor resistance variant populations in clinical samples early in therapy to allow for customizable patient treatment and to track variant populations' progress through time. The present invention addresses this need by providing methods to detect heteroresistance in TB patient samples at low resistance levels using multiple target loci amplification and sequencing with clinically relevant next generation technology. This translates to a significant increase in the level of detection sensitivity as compared to other methods and an enhanced ability to treat patients with effective therapies against the resistant pathogen populations.